A number of methods are known for selectively targeting cells in a patient for delivery of diagnostic or therapeutic agents. Selective targeting has led to the introduction of various diagnostic agents for visualization of tissues, such as contrast agents useful in Magnetic Resonance Imaging (MRI), radiodiagnostic compositions, and the like. Introduction of therapeutic agents, such as compositions for radiotherapy or for neutron capture therapy, compositions for chemotherapy, various proteins, peptides, and nucleic acids, protein toxins, antisense oligonucleotides, liposomes, analgesics, antibiotics, antihypertensive agents, antiviral agents, antihistamines, expectorants, vitamins, plasmids, and the like, has also been demonstrated.
Folate conjugates have been used for the selective targeting of cell populations expressing folate receptors or other folate binding proteins to label or deliver bioactive compounds to such cells. The relative populations of these receptors and binding proteins have been exploited in achieving selectivity in the targeting of certain cells and tissues, such as the selective targeting of tumors expressing elevated levels of high-affinity folate receptors. The following publications, the disclosures of which are incorporated herein by reference, illustrate the nature and use of folate conjugates for diagnosis or delivery of biologically significant compounds to selected cell populations in patients in need of such diagnosis or treatment:                (a) Leamon and Low, “Cytotoxicity of Momordin-folate Conjugates in Cultured Human Cells” in J. Biol. Chem., 1992, 267, 24966-24967.        (b) Leamon et al., “Cytotoxicity of Folate-pseudomonas Exotoxin Conjugates Towards Tumor Cells” in J. Biol. Chem., 1993, 268, 24847-24854.        (c) Lee and Low, “Delivery of Liposomes into Cultured Kb Cells via Folate Receptor-mediated Endocytosis” in J. Biol. Chem., 1994, 269, 3198-3204.        (d) Wang et al., “Delivery of Antisense Oligonucleotides Against the Human Epidermal Growth Factor Receptor into Cultured Kb Cells with Liposomes Conjugated to Folate via Polyethyleneglycol” in Proc. Natl. Acad. Sci. USA., 1995, 92, 3318-3322.        (e) Wang et al., “Synthesis, Purification and Tumor Cell Uptake of Ga-67-deferoxamine-folate, a Potential Radiopharmaceutical for Tumor Imaging” in Bioconj. Chem., 1996, 7, 56-63.        (f) Leamon et al., “Delivery of Macromolecules into Living Cells: a Method That Exploits Folate Receptor Endocytosis” in Proc. Natl. Acad. Sci., U.S.A., 1991, 88, 5572-5576.        (g) Krantz et al., “Conjugates of Folate Anti-Effector Cell Antibodies” in U.S. Pat. No. 5,547,668.        (h) Wedeking el al., “Metal Complexes Derivatized with Folate for Use in Diagnostic and Therapeutic Applications” in U.S. Pat. No. 6,093,382.        (i) Low et al., “Method for Enhancing Transmembrane Transport of Exogenous Molecules” in U.S. Pat. No. 5,416,016.        (j) Miotti et al., “Characterization of Human Ovarian Carcinoma-Associated Antigens Defined by Novel Monoclonal Antibodies with Tumor-Restricted Specificity” in Int. J. Cancer, 1987, 39, 297-303.        (k) Campell et al., “Folate-Binding Protein is a Marker for Ovarian Cancer” in Cancer Res., 1991, 51, 5329-5338.        (l) Jansen et al., “Identification of a Membrane-Associated Folate-Binding Protein in Human Leukemic CCRF-CEM Cells with Transport-Related Methotrexate Resistance” in Cancer Res., 1989, 49, 2455-2459.        
Multiple types of folate recognition sites present on cells, such as α-folate receptors, β-folate receptors, folate binding proteins, and the like, have been shown to recognize and bind the conjugates described above. The primary pathway for entry of folate derivatives into cells is through a facilitated transport mechanism mediated by a membrane transport protein. However, when folate is covalently conjugated to certain small molecules and macromolecules, the transport system can fail to recognize the folate molecule.
Advantageously, in addition to the facilitated transport protein, some cells possess a second membrane-bound receptor, folate binding protein (FBP), that allows folate uptake via receptor-mediated endocytosis. At physiological plasma concentrations (nanomolar range), folic acid binds to cell surface receptors and is internalized via an endocytic process. Receptor-mediated endocytosis is the movement of extracellular ligands bound to cell surface receptors into the interior of the cells through invagination of the membrane, a process that is initiated by the binding of a ligand to its specific receptor. The uptake of substances by receptor-mediated endocytosis is a characteristic ability of some normal, healthy cells such as macrophages, hepatocytes, fibroblasts, reticulocytes, and the like, as well as abnormal or pathogenic cells, such as tumor cells. Notably, folate binding proteins involved in endocytosis are less sensitive to modification of the folate molecule than the membrane transport proteins, and often recognize folate conjugates. Both targeting and uptake of conjugated diagnostic and therapeutic agents are enhanced.
Following endosome acidification, the folate receptor changes conformation near its ligand-binding domain and releases the folic acid molecule. Folate receptors are known to recycle back to the membrane surface for additional rounds of ligand-mediated internalization. However, a significant fraction of the internalized receptor-folic acid complex has been shown to return back to the cell surface shortly after endocytosis. This suggests that the acid-triggered ligand release mechanism does not proceed to completion, at least after the first round of internalization (Kamen et al., 1988, J. Biol. Chem. 263, 13602-13609).
Pteroic acid, which is essentially folic acid lacking the distal glutamyl residue (FIG. 1), does not bind to the high-affinity folate receptor to any appreciable extent (Kamen et al., 1986, Proc. Natl. Acad. Sci., USA. 83, 5983-5987); in fact, 2 μM pteroic acid (100-fold excess) had absolutely no effect on the binding of folate to the folate receptor. Thus, the glutamyl residue of folic acid, or some portion thereof, was generally thought to be required for efficient, specific receptor recognition. However, recent studies have revealed that the glutamyl residue of folic acid could be replaced with a lysyl residue without disturbing the binding affinity of the ligand (McAlinden et al., 1991, Biochemistry 30, 5674-5681; Wu et al., 1997, J. Membrane Biol. 159, 137-147), that the glutamyl residue can be replaced with a glycyl residue without substantially altering cellular uptake, and that no selective isomeric (i.e., α-glutamyl vs. γ-glutamyl) conjugation requirement necessarily exists (Leamon et al., J. Drug Targeting 7:157-169 (1999); Linder et al., J. Nuclear Med. 41(5):470 Suppl. 2000).
Efforts to improve the selectivity of targeting or increase the diversity of the agents delivered to the cell or tissue have been hampered by a number of complications, including the complex syntheses required for the preparation of these conjugates. Such synthetic schemes are not only time consuming, but may also preclude the use of certain conjugates due to synthetic incompatibilities. A folic acid analog capable of expanding the number or diversity of agents, via the conjugates of such agents and these folic acid analogs, presentable to target cells would be advantageous.